Selectivity is an important factor for a successful chromatographic separation. Common stationary phases for liquid chromatography, such as reversed-phase (RP), ion-exchange (IEX) and normal phase (NP) chromatography are frequently characterized by limited selectivity necessitating multiple analyses for a single sample.
Ion-exchange liquid chromatography (IEX-LC) is used to separate ionic or ionizable compounds. Despite its important role in the separation of proteins, nucleic acids, and inorganic ions, ion-exchange chromatography is rarely used for the analysis of hydrophobic, e.g., small organic molecules, partially due to the lack of hydrophobic retention.
Ion-pairing liquid chromatography is a powerful tool for manipulating the selectivity of a separation. It involves the addition of an ion-pairing reagent to the mobile phase to promote the formation of charged analytes. These reagents are comprised of an alkyl chain with an ionizable terminus. The columns used in ion-pairing chromatography are typically reverse phase (RP) columns (e.g. C18 or C8). Analytes with an opposite charge to the ion pairing reagent are retained longer, and the retention of analytes with the same charge as the pairing reagent is reduced. However, the retention of neutral analytes is nearly unaffected by the use of an ion-pairing reagent. In addition, ion-pairing chromatography often requires a dedicated column and long equilibration times. Further, the mobile phase is typically incompatible with mass-spectroscopy.
Mixed-mode chromatography combines aspects of ion-exchange and reverse-phase chromatography. The columns for mixed-mode chromatography combine both hydrophobic and ion-exchange functionalities and facilitate adjustable selectivity for a variety of different molecules. Hydrophilic and ionizable compounds that are difficult to separate using reverse-phase columns can frequently be resolved using mixed-mode resins. Generally, mixed-mode stationary phases incorporate either reversed-phase and anion-exchange capabilities or reversed-phase and cation-exchange capabilities and typically do not provide sufficient selectivity to retain and separate cations, anions and neutral analytes within a single analysis.
Packing materials bearing amphoteric or zwitterionic functionalities are known. However these materials are used with either anion-exchange or cation-exchange conditions, but do not provide both of these capabilities simultaneously. In these stationary phases, positive and negative charges are not sufficiently separated and tend to neutralize one another. Hence, a need exists for stationary phases that provide both cation-exchange and anion-exchange capabilities on the same solid support. It is further desirable to provide trimodal stationary phases with anion-exchange, cation-exchange and reverse-phase functionalities in order to retain and separate cations, anions and uncharged molecules within a single analysis. The present invention addresses these and other needs.